Assaying bodily fluids such as blood for levels of various organic molecules is useful in the treatment of diseased states. For example, diabetes mellitus is a disease characterized by poor regulation of blood glucose levels. The traditional treatments for mild forms of this disease, including adult onset diabetes, have included diet and exercise. More severe forms, however, require administration of insulin. One of the drawbacks of administering insulin is the possibility of insulin shock, caused by rapid decrease in blood glucose levels (glucose imbalance) due to unintended over medication. Insulin shock is, however, only the most severe manifestation of glucose imbalance. The consequences of chronic glucose imbalance (both over and under medication) are well documented and include damage to blood vessels and various body organs. Blindness is common, as is the loss of circulation in the extremities.
Accurate measurement of blood glucose levels would enable the patient to modulate insulin dosage and avoid the effects of chronic glucose imbalance. One example of a prior art attempt at glucose measurement is a glucose sensor as disclosed in U.S. Pat. No. 3,542,662. In this device, an enzyme-containing membrane is disposed between a fluid being assayed and a first oxygen sensor electrode. A similar membrane not containing enzyme is disposed between the fluid and a second reference oxygen sensor electrode. A certain portion of the oxygen diffusing through the enzyme-containing membrane is consumed by equimolar reaction with glucose catalyzed by the enzyme and is therefore unavailable for detection by the first oxygen sensor electrode. The second, reference oxygen sensor electrode, in which the membrane does not include enzyme, determines the concentration of oxygen that would have been detected had not the enzyme-promoted reaction occurred. The difference in oxygen detected by the two electrodes is indicative of the glucose concentration.
A problem with this device is that the levels of oxygen and glucose in the blood are less than stoichiometric. In particular, the amount of oxygen is less than that needed to convert all the glucose. Thus the sensor can become oxygen limited and not respond accurately at high glucose concentrations.
In order to bring the levels of glucose and oxygen to stoichiometric balance and thus create a device that gives accurate results over the complete range of glucose concentrations found in blood, it has been proposed to design sensors that reduce the amount of glucose reaching the enzyme layer relative to oxygen. This could be accomplished in theory by providing a membrane layer which is significantly more permeable to oxygen than glucose. U.S. Pat. No. 4,650,547, described more fully hereinbelow, provides a general description of this concept.
Implementing this approach has heretofore been difficult, however, due to the prior art's inability to precisely and reproducibly control the permeability of the membrane. Without such precise control, the glucose flux reaching the enzyme layer may not be sufficiently attenuated. Problems can also arise due to the presence of interferant molecules, e.g. ascorbate and urate. The determination of creatinine levels, which is used to measure renal function, is an example of an analyte that requires removal of these interferants.
U.S. Pat. No. 4,933,048 relates to water permeable, ion impermeable membranes microfabricated over a hydrogel layer leaving an opening for ion exchange. FIG. 2 of the '048 patent illustrates a structure where the opening is formed by having the hydrogel layer extend beyond the ion impermeable layer. Alternatively, the ion impermeable layer can cover the entire hydrogel layer with holes formed beyond the perimeter of the underlying electrode (column 7, line 1). Holes can be formed by laser perforation or other methods. The aperture is formed at a distance from the electrode and the function of the small opening is to provide a low impedance electrolytic junction.
Glucose sensors using non-microfabricated or "macro" electrodes are known. See, for example, Fischer, U. and Abel, P., Transactions of the American Society of Artificial Internal Organs 1982, 28, 245-248 (Fischer et al.); Rehwald, W., Pflugers Archiv 1984, 400, 348-402; U.S. Pat. Nos. 4,484,987; 4,515,584; and 4,679,562; and UK Patent Application 2,194,843. However, no aspect of thin-film processing is described in these documents.
Fischer et al. discloses a non-microfabricated glucose sensor with a Teflon.RTM. membrane which is mechanically perforated. Glucose can only enter through the perforation whereas oxygen can pass through the Teflon.RTM., thus adjusting the stoichiometry in the enzyme layer and linearizing the response. There is no teaching of optimizing or controlling the dimensions of the perforation. The Fischer et al. document is also silent on the use of microfabrication. East German patent DD 282527 appears to correspond to this publication but does not name Fischer as an inventor.
U.S. Pat. No. 4,484,987 relates to a linearized glucose sensor based on the concept of providing a layer with hydrophobic regions in a hydrophilic matrix where glucose can permeate the latter but not the former, and oxygen can permeate both regions (see description of FIG. 1 thereof. In an alternative embodiment, shown in FIG. 4, a hydrophobic layer includes spaced small openings through which glucose molecules can pass. However, the '987 patent provides no teaching of how the dimensions or location of the openings are controlled and is silent on microfabrication.
U.S. Pat. No. 4,650,547 discloses a glucose sensor where a hydrophobic gas permeable membrane is placed over a hydrophilic enzyme-containing layer, where only the perimeter or peripheral edge thickness surface of the hydrophilic layer is exposed to the sample (FIG. 5). Glucose can only enter the hydrophilic layer at the perimeter and diffuse parallel to the plane of the layer, whereas oxygen can be supplied across the entire surface of the hydrophobic layer (column 6, line 3).
Anal Chem 57, 2351, 1985 provides teaching for making a related cylindrical device where the gap between a platinum wire electrode and a gas permeable cylindrical coating is filled with an enzyme gel. There is no teaching, however, of microfabrication. U.S. Pat. No. 4,890,620 relates to a similar structure and method based on a differential measurement with a pair of sensors. An implantable version is disclosed in U.S. Pat. No. 4,703,756.
Regarding lactate and creatinine, there is comparatively little sensor literature. In Clin. Chem. 29, 51, 1983, there is proposed an amperometric creatinine sensor using three enzymes coupled to the production of hydrogen peroxide. This document also includes a differential measurement where one sensor measures creatine and the other measures creatine plus creatinine. The sensors are made using a cellulose acetate--glutaraldehyde method. Anal Chem 67, 2776, 1995 teaches electropolymerization to immobilize the creatinine enzymes onto an electrode. A poly(carbamoyl)sulphonate hydrogel is used in Anal Chim Acta 325, 161, 1996. None of the above documents teaches the use of microfabrication. Microdispensing to establish enzyme gel layers onto electrodes made by microfabrication is, however, disclosed in Anal Chim Acta 319, 335, 1996.
Despite the recent and significant advances in analyte sensors exemplified by U.S. Pat. Nos. 5,200,052 and 5,096,669, there remains a need in the art for improved microfabrication techniques and greater control of analyte flux. There is further a need in the art for reducing or eliminating the effect of interferant molecules on sensor measurement.
The measurement of glucose with a microfabricated sensor, described in U.S. Pat. No. 5,200,051, assigned to i-STAT Corporation, uses a thin contiguous analyte attenuation (AA) layer made from a silicone copolymer to cover an enzyme layer. It provides a membrane that is freely permeable to oxygen but is poorly permeable to glucose. This enables a linear response over the full range of glucose concentrations found in blood. As the '051 patent makes clear, oxygen is required in stoichiometric amounts to sustain the enzymatic reaction, despite the low levels generally present in blood. Using this membrane achieves this goal. The '051 patent includes a discussion of the general properties of a microfabricated analyte attenuation layer at column 12, beginning at line 57, with a more detailed description beginning at column 38, line 19. The etch process for the AA layer is discussed beginning at column 58, line 5. Structures with open perimeters for measuring glucose are illustrated in FIGS. 7A & 8A of the '051 patent, however, glucose transport occurs through a polysiloxane copolymer layer.
Wholly microfabricated sensors, that is, sensors which are uniformly mass-produced by thin-film techniques and micro-manufacturing methods, had not demonstrated utility in a clinical setting prior to the '051 patent. The '051 patent showed that the degree of complexity involved with the mass production of commercially viable biosensors was much more formidable than those persons of ordinary skill in the art once perceived. Of major concern was the compatibility of inherently harsh physical and chemical processes associated with the then existing commercial microfabrication manufacturing methods.
An article by Eleccion (Eleccion, M. Electronics 1986, Jun. 2, 26-30) describes the then current state of the art with regard to microsensors and makes brief references to active areas of research including the detection of specific ions, gases, and biological materials. Progress in the area of field effect transistors (FETs) is noted and problems and limitations with present manufacturing methods are discussed.
It is also important to note that in current clinical settings medical practitioners commonly request analyses of one or more components of a complex biological fluid such as whole blood. Currently, such analyses require a certain amount of processing of whole blood, such as filtration and centrifugation, to avoid contamination of the instruments or to simplify subsequent measurements. Frequently, blood samples are sent to a remote central laboratory where the analyses are performed. Patients and physicians are thus deprived of valuable information, which, in most cases, is not available for hours, sometimes days. Clearly, substantial advantages could be envisaged if analyses on undiluted samples could be carried out and if instruments or sensors were available perform real-time measurements. This can now be achieved using the point-of-care blood analysis system described in U.S. Pat. No. 5,096,669 (assigned to i-STAT Corporation).
Despite the recent and significant advances in chemical sensor technology as exemplified by U.S. Pat. Nos. 5,200,051 and 5,096,669, there remains a need in the art for improved microfabrication techniques and greater control of analyte flux. There is further a need in the art for reducing or eliminating the effect of interferant molecules on sensor measurement.